Requirement for intron structures in activating the locus.

During differentiation of CD4CD8 double-positive (DP) thymocytes into the CD4CD8 single-positive (CD8SP) thymocytes committed to the cytotoxic T cell lineage, transcription is temporally terminated after positive selection and is subsequently reinitiated, a process known as coreceptor reversal. Despite the identification of a transcriptional enhancer in the gene that directs reporter transgene expression specifically in CD8SP thymocytes, the molecular mechanisms controlling reactivation of the gene are not fully understood. Here, we show that, after positive selection, hCD2 reporter expression from the locus, which was generated by insertion of hCD2 cDNA into the first exon of the gene, requires the incorporation of intron sequences into the transcript. The presence of polyadenylation signals after hCD2 cDNA inhibited hCD2 expression in mature CD8 T cells, whereas hCD2 expression in DP thymocytes recapitulated the expression. Incorporation of the endogenous short intron structure and heterologous intron structure of the locus restored hCD2 expression in mature CD8 T cells in a variegated manner. Interestingly, stage-specific DNA demethylation was impaired in reporter alleles that failed to express hCD2 in CD8 T cells, and intron sequences lacking RNA splicing signals still restored hCD2 expression. These observations indicate that "intron-mediated enhancement" is involved in a stage-specific reactivation of the locus harboring hCD2 cDNA. However, the gene was transcribed in mature CD8 T cells, albeit at a lower level, from a mutant locus lacking intron structures, suggesting that protein-coding sequences in transcripts affect sensitivity to intron-mediated enhancement.